Label-Free Quantification of Molecular Interaction in Live Red Blood Cells by Tracking Nanometer Scale Membrane Fluctuations.

Molecular interactions in live cells play an important role in both cellular functions and drug discovery. Current methods for measuring binding kinetics involve extracting the membrane protein and labeling, while the in situ quantification of molecular interaction with surface plasmon resonance (SPR) imaging mainly worked with fixed cells due to the micro-motion related noises of live cells. Here, an optical imaging method is presented to measure the molecular interaction with live red blood cells by tracking the nanometer membrane fluctuations. The membrane fluctuation dynamics are measured by tracking the membrane displacement during glycoprotein interaction. The data are analyzed with a thermodynamic model to determine the elastic properties of the cell observing reduced membrane fluctuations under fixatives, indicating cell fixations affect membrane mechanical properties. The binding kinetics of glycoprotein to several lectins are obtained by tracking the membrane fluctuation amplitude changes on single live cells. The binding kinetics and strength of different lectins are quite different, indicating the glycoproteins expression heterogeneity in single cells. It is anticipated that the method will contribute to the understanding of mechanisms of cell interaction and communication, and have potential applications in the mechanical assessment of cancer or other diseases at the single-cell level, and screening of membrane protein targeting drugs.

Gradient-Based Colorimetric Array Sensor for Continuous Monitoring of Multiple Gas Analytes.

Colorimetry is widely used in chemical sensing due to its high sensitivity and high selectivity. However, most colorimetric sensors are one-time use because the color-producing reactions or bindings are usually irreversible. In addition, traditional colorimetric sensors like the detection tubes are bulky and packed individually, making parallel sensing of multiple analytes difficult. Here, we demonstrate a gradient-based colorimetric array sensor (GCAS) to overcome these limitations. Different colorimetric sensing elements are inkjet-printed as parallel straight lines on a porous substrate. Lateral transport of analytes across the substrate creates color gradients on the sensing elements. The color gradients shift along the transport direction over time, and GCAS tracks the gradient shifts and converts them into analyte concentrations in real time. Using a low-cost complementary metal-oxide semiconductor imager, we show detection of three air pollutants using a single GCAS chip and 24 h continuous monitoring of ambient ozone.

Probing Single-Molecule Binding Event by the Dynamic Counting and Mapping of Individual Nanoparticles.

Measuring binding processes at the single-molecule level underpin significant functions in understanding biological events. Single-nanoparticle imaging techniques are providing a new concept for mapping the heterogeneous behaviors and characterizations of individual dynamics such as molecule-molecule interactions. Here, we develop the optical imaging techniques for directly counting and monitoring the binding and motion events of single nanoparticles linked to the substrate via the specific and reversible interactions between biomolecules. The one-step digital immunoassay realizes the biomolecular detection based on dynamic counting of the single nanoparticle binding event to substrate with the bright-field imaging. The detection limit achieves 8.4 pg/mL for procalcitonin with detection time of 14 min. Meanwhile, we map the accurate trajectory of single nanoparticle switching between different target molecules among the x-y plane with the total internal reflection imaging technique, which reveals the spatial coordinates of single target molecules on the substrate surface with high spatial and temporal resolutions.

Total Iron Measurement in Human Serum With a Novel Smartphone-Based Assay.

Background: Abnormally low or high blood iron levels are common health conditions worldwide and can seriously affect an individual's overall well-being. A low-cost point-of-care technology that measures blood iron markers with a goal of both preventing and treating iron-related disorders represents a significant advancement in medical care delivery systems. Methods: A novel assay equipped with an accurate, storable, and robust dry sensor strip, as well as a smartphone mount and (iPhone) app is used to measure total iron in human serum. The sensor strip has a vertical flow design and is based on an optimized chemical reaction. The reaction strips iron ions from blood-transport proteins, reduces Fe(III) to Fe(II), and chelates Fe(II) with ferene, with the change indicated by a blue color on the strip. The smartphone mount is robust and controls the light source of the color reading App, which is calibrated to obtain output iron concentration results. The real serum samples are then used to assess iron concentrations from the new assay, and validated through intra-laboratory and inter-laboratory experiments. The intra-laboratory validation uses an optimized iron detection assay with multi-well plate spectrophotometry. The inter-laboratory validation method is performed in a commercial testing facility (LabCorp). Results: The novel assay with the dry sensor strip and smartphone mount, and App is seen to be sensitive to iron detection with a dynamic range of 50 - [Formula: see text]/dL, sensitivity of 0.00049 a.u/[Formula: see text]/dL, coefficient of variation (CV) of 10.5%, and an estimated detection limit of [Formula: see text]/dL These analytical specifications are useful for predicting iron deficiency and overloads. The optimized reference method has a sensitivity of 0.00093 a.u/[Formula: see text]/dL and CV of 2.2%. The correlation of serum iron concentrations (N = 20) between the optimized reference method and the novel assay renders a slope of 0.95, and a regression coefficient of 0.98, suggesting that the new assay is accurate. Last, a spectrophotometric study of the iron detection reaction kinetics is seen to reveal the reaction order for iron and chelating agent. Conclusion: The new assay is able to provide accurate results in intra- and inter- laboraty validations, and has promising features of both mobility and low-cost manufacturing suitable for global healthcare settings.

Integrating Electrochemical and Colorimetric Sensors with a Webcam Readout for Multiple Gas Detection.

Multisensor detectors have merits of low cost, compact size, and capability of supplying accurate and reliable information otherwise hard to obtain by any single sensors. They are therefore highly desired in various applications. Despite the advantages and needs, they face great challenges in technique especially when integrating sensors with different sensing principles. To bridge the gap between the demand and technique, we here demonstrated an integration of electrochemical and colorimetric sensors with a webcam readout for multiple gas detection. Designed with two parallel gas channels but independent sensor cells, the dual-sensor detector could simultaneously detect each gas from their gas mixture by analysis of the group photo of the two sensors. Using Ag electro-dissolution as reporter, the bipolar electrochemical sensor achieved quantitative analysis for the first time thanks to application of pulse voltage. The sacrificed Ag layer used in the bipolar electrochemical (EC) sensor was recycled from CD, which further decreased the sensor cost and supplied a new way of CD recycling. The EC O2 sensor response, edge displacement of Ag layer due to electrochemical dissolution, has a linear relationship with O2 concentration ranging from 0 to 30% and has good selectivity to common oxidative gases. The colorimetric NO2 sensor linearly responded to NO2 concentrations ranging from 0 to 230 ppb with low detection limit of 10 ppb, good selectivity, and humidity tolerance. This integration method could be extended to integrating other gas sensors.

Time-Resolved Digital Immunoassay for Rapid and Sensitive Quantitation of Procalcitonin with Plasmonic Imaging.

Timely diagnosis of acute diseases improves treatment outcomes and saves lives, but it requires fast and precision quantification of biomarkers. Here, we report a time-resolved digital immunoassay based on plasmonic imaging of binding of single nanoparticles to biomarkers captured on a sensor surface. The real-time and high contrast of plasmonic imaging lead to fast and precise counting of the individual biomarkers over a wide dynamic range. We demonstrated the detection principle, evaluated the performance of the method using procalcitonin (PCT) as an example, and achieved a limit of detection of ∼2.8 pg/mL, dynamic range of 4.2-12500 pg/mL, for a total detection time of ∼25 min.

Optical Tracking of Nanometer-Scale Cellular Membrane Deformation Associated with Single Vesicle Release.

Exocytosis involves interactions between secretory vesicles and the plasma membrane. Studying the membrane response is thus critical to understand this important cellular process and to differentiate different mediator release patterns. Here we introduce a label-free optical imaging method to detect the vesicle-membrane-interaction-induced membrane deformation associated with single exocytosis in mast cells. We show that the plasma membrane expands by a few tens of nanometers accompanying each vesicle-release event, but the dynamics of the membrane deformation varies from cell to cell, which reflect different exocytosis processes. Combining the temporal and spatial information allows us to resolve complex vesicle-release processes, such as two vesicle-release events that occur closely in time and location. Simultaneous following a vesicle release with fluorescence and membrane deformation tracking further allows us to determine the propagation speed of the vesicle-release-induced membrane deformation along the cell surface, which has an average value of 5.2 ± 1.8 µm/s.

Gradient-Based Colorimetric Sensors for Continuous Gas Monitoring.

Colorimetry detects a color change resulted from a chemical reaction or molecular binding. Despite its widespread use in sensing, continuous monitoring of analytes with colorimetry is difficult, especially when the color-producing reaction or binding is irreversible. Here, we report on a gradient-based colorimetric sensor (GCS) to overcome this limitation. Lateral transport of analytes across a colorimetric sensor surface creates a color gradient that shifts along the transport direction over time, and GCS tracks the gradient shift and converts it into analyte concentration in real time. Using a low cost complementary metal-oxide semiconductor imager and imaging processing algorithm, we show submicrometer gradient shift tracking precision and continuous monitoring of ppb-level ozone.

Measuring the Spin-Polarization Power of a Single Chiral Molecule.

The electronic spin filtering capability of a single chiral helical peptide is measured. A ferromagnetic electrode source is employed to inject spin-polarized electrons in an asymmetric single-molecule junction bridging an α-helical peptide sequence of known chirality. The conductance comparison between both isomers allows the direct determination of the polarization power of an individual chiral molecule.

Label-Free Imaging of Histamine Mediated G Protein-Coupled Receptors Activation in Live Cells.

G protein-coupled receptors (GPCRs) are the largest protein family for cell signal transduction, and most of them are crucial drug targets. Conventional label-free assays lack the spatial information to address the heterogeneous response from single cells after GPCRs activation. Here, we reported a GPCRs study in live cells using plasmonic-based electrochemical impedance microscopy. This label-free optical imaging platform is able to resolve responses from individual cells with subcellular resolution. Using this platform, we studied the histamine mediated GPCRs activation and revealed spatiotemporal heterogeneity of cellular downstream responses. Triphasic responses were observed from individual HeLa cells upon histamine stimulation. A quick peak P1 in less than 10 s was attributed to the GPCRs triggered calcium release. An inverted P2 phase within 1 min was attributed to the alternations of cell-matrix adhesion after the activation of Protein Kinase C (PKC). The main peak (P3) around 3-6 min after the histamine treatment was due to dynamic mass redistribution and showed a dose-dependent response with a half-maximal effective concentration (EC50) of 3.9 ± 1.2 µM. Heterogeneous P3 responses among individual cells were observed, particularly at high histamine concentration, indicating diverse histamine H1 receptor expression level in the cell population.

Imaging Local Heating and Thermal Diffusion of Nanomaterials with Plasmonic Thermal Microscopy.

Measuring local heat generation and dissipation in nanomaterials is critical for understanding the basic properties and developing applications of nanomaterials, including photothermal therapy and joule heating of nanoelectronics. Several technologies have been developed to probe local temperature distributions in nanomaterials, but a sensitive thermal imaging technology with high temporal and spatial resolution is still lacking. Here, we describe plasmonic thermal microscopy (PTM) to image local heat generation and diffusion from nanostructures in biologically relevant aqueous solutions. We demonstrate that PTM can detect local temperature change as small as 6 mK with temporal resolution of 10 µs and spatial resolution of submicrons (diffraction limit). With PTM, we have successfully imaged photothermal generation from single nanoparticles and graphene pieces, studied spatiotemporal distribution of temperature surrounding a heated nanoparticle, and observed heating at defect sites in graphene. We further show that the PTM images are in quantitative agreement with theoretical simulations based on heat transport theories.

Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging.

Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 10(5) M(-1) s(-1) and (1.4 ± 0.5) × 10(-4) s(-1), respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/µm(2) with an estimation of 5 × 10(5) EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines (A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.

Measuring Binding Kinetics of Antibody-Conjugated Gold Nanoparticles with Intact Cells.

Antibody-conjugated nanomaterials have attracted much attention because of their applications in nanomedicine and nanotheranostics, and amplification of detection signals. For many of these applications, the nanoconjugates must bind with a cell membrane receptor (antigen) specifically before entering the cells and reaching the final target, which is thus important but not well understood. Here, a plasmonic imaging study of the binding kinetics of antibody-conjugated gold nanoparticles with antigen-expressing cells is presented, and the results are compared with that of the nanoparticle-free antibody. It is found that the nanoconjugates can significantly affect the binding kinetics compared with free antibody molecules, depending on the density of the antibody conjugated on the nanoparticles, and expressing level of the antigen on the cell membrane. The results are analyzed in terms of a transition from monovalent binding model to a bivalent binding model when the conjugation density and expressing level increase. These findings help optimize the design of functional nanomaterials for drug delivery and correct interpretation of data obtained with nanoparticle signal amplification.

Real-time monitoring of phosphorylation kinetics with self-assembled nano-oscillators.

Phosphorylation is a post-translational modification that is involved in many basic cellular processes and diseases, but is difficult to detect in real time with existing technologies. A label-free detection of phosphorylation is reported in real time with self-assembled nano-oscillators. Each nano-oscillator consists of a gold nanoparticle tethered to a gold surface with a molecular linker. When the nanoparticle is charged, the nano-oscillator can be driven into oscillation with an electric field and detected with a plasmonic imaging approach. The nano-oscillators measure charge change associated with phosphorylation of peptides attached onto a single nanoparticle, allowing us to study the dynamic process of phosphorylation in real time without antibodies down to a few molecules, from which Michaelis and catalytic rate constants are determined.

Detection of molecular binding via charge-induced mechanical response of optical fibers.

We report a charge sensitive optical detection technique for label-free study of molecular interactions. Traditional label-free optical detection techniques largely rely on the detection of the mass of a molecule, which are insensitive to small molecules. In contrast, the present technique detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, the technique is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. Using the technique, we have detected 0.2 nM anti-BSA and 15 µM anti-cancer drug (imatinib) with an enzyme modified surface. The achieved effective charge detection limit is ~0.25 electron charge/µm2, corresponding to ~0.3 fg/mm2 for imatinib, which is orders of magnitude better than traditional label-free optical detection methods.

In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance.

Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

Measurement of small molecule binding kinetics on a protein microarray by plasmonic-based electrochemical impedance imaging.

We report on a quantitative study of small molecule binding kinetics on protein microarrays with plasmonic-based electrochemical impedance microscopy (P-EIM). P-EIM measures electrical impedance optically with high spatial resolution by converting a surface charge change to a surface plasmon resonance (SPR) image intensity change, and the signal is not scaled to the mass of the analyte. Using P-EIM, we measured binding kinetics and affinity between small molecule drugs (imatinib and SB202190) and their target proteins (kinases Abl1 and p38-α). The measured affinity values are consistent with reported values measured by an indirect competitive binding assay. We also found that SB202190 has weak bindings to ABL1 with KD > 10 µM, which is not reported in the literature. Furthermore, we found that P-EIM is less prone to nonspecific binding, a long-standing issue in SPR. Our results show that P-EIM is a novel method for high-throughput measurement of small molecule binding kinetics and affinity, which is critical to the understanding of small molecules in biological systems and discovery of small molecule drugs.

Real-time monitoring biomarker expression of carcinoma cells by surface plasmon resonance biosensors.

A novel surface plasmon resonance (SPR) biosensor which is capable of monitoring proteomic biomarker secretion from living cells is reported here. Vascular endothelial growth factor (VEGF) secretion from living SKOV-3 ovarian cancer cells was measured for concept demonstration.

Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy.

Electrochemical impedance spectroscopy is a crucial tool for the detection and study of various biological substances, from DNA and proteins to viruses and bacteria. It does not require any labelling species, and methods based on it have been developed to study cellular processes (such as cell spreading, adhesion, invasion, toxicology and mobility). However, data have so far lacked spatial information, which is essential for investigating heterogeneous processes and imaging high-throughput microarrays. Here, we report an electrochemical impedance microscope based on surface plasmon resonance that resolves local impedance with submicrometre spatial resolution. We have used an electrochemical impedance microscope to monitor the dynamics of cellular processes (apoptosis and electroporation of individual cells) with millisecond time resolution. The high spatial and temporal resolution makes it possible to study individual cells, but also resolve subcellular structures and processes without labels, and with excellent detection sensitivity (~2 pS). We also describe a model that simulates cellular and electrochemical impedance microscope images based on local dielectric constant and conductivity.